The human type I iodothyronine 5'-deiodinase gene encodes a member of the family of selenocysteine-containing deiodinases. These enzymes catalyze the activation of the prohormone thyroxine to 3,3',5-triiodothyronine or the degradation of thyroxine and triiodothyronine to inactive metabolites. Here we report the isolation of two genomic type I 5'-deiodinase clones from a chromosome 1-specific gridded cosmid library, the localization of the gene to chromosome 1p32-p33 by fluorescence in situ hybridization, and the determination of the complete structure of the 17.5-kb gene.